A novel polypeptide and its uses

Abstract

This invention provides a polypeptide whose amino acid sequence is shown in SEQ ID NO.1. Specifically, the sequence of the polypeptide is: MTSRQEDASGKKTTEGAKFQGS. This invention also provides a DNA molecule encoding the polypeptide of claim 1. This invention further provides a recombinant vector containing the above-mentioned DNA molecule. This invention also provides the use of the above-mentioned polypeptide in the preparation of a medicament for treating myocardial fibrosis. This invention also provides a pharmaceutical composition in which the above-mentioned polypeptide is the active ingredient. Experiments have shown that this invention can alleviate myocardial fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a polypeptide, specifically a novel polypeptide and its uses. Background Technology

[0002] Heart failure (HF) is the end stage of various heart diseases, severely impacting patients' quality of life and leading to malignant arrhythmias and even sudden death. Due to global population aging and improved treatment effectiveness and survival rates, HF's prevalence is increasing. Recent projections indicate that the prevalence of HF will increase by approximately 46% from 2012 to 2030, making it a serious global public health problem. Current clinical treatments for HF primarily include drug therapy, interventional procedures, and heart transplantation; however, these methods all have limitations, resulting in a 5-year mortality rate of approximately 50% for chronic HF patients. Therefore, finding safer and more effective strategies for the prevention and treatment of HF is of great significance.

[0003] Myocardial fibrosis leads to systolic and diastolic dysfunction under many cardiac pathophysiological conditions. Existing studies have found that: ① Alamandine alleviates heart failure and reduces cardiac fibrosis by inhibiting the increase of collagen I, α-SMA, and TGF-β in neonatal rat cardiac fibroblasts induced by oxygen-glucose deprivation (OGD). ② HIMF induces myocardial fibrosis by activating the MAPK and CaMKII-STAT3 pathways to mediate paracrine effects from cardiomyocytes to fibroblasts. ③ AMFR catalyzes the ubiquitination of FAM134B at k27 and k33 linkages, enhancing endoplasmic reticulum phagocytic flux, thereby inhibiting phosphorylation of downstream targets of mTORC1 such as S6K1 and 4E-BP, thus inhibiting myocardial fibrosis.

[0004] The beneficial effects of long-term, regular exercise on the cardiovascular system have become a consensus in the field. In recent years, with the development of relevant clinical research, the good preventive and therapeutic effects of exercise on heart failure have received increasing attention. Studies have shown that regular and moderate exercise can reduce myocardial oxidative stress, activate VEGFR, increase myocardial angiogenesis, prevent cardiac remodeling after myocardial infarction, and improve myocardial diastolic and systolic function. This not only effectively reduces the risk of heart failure but also reduces all-cause mortality and hospitalization rates in heart failure patients and significantly improves patient prognosis. Currently, more and more studies have found that exercise, as a safe physiological stimulus, has a good preventive and therapeutic effect on heart failure.

[0005] Studies have shown that peptides, due to their unique biological properties, have significant value in regulating human physiological functions and in clinical diagnosis and treatment, and have become a popular direction in biomedical research and clinical studies. As an ideal candidate molecule for drug development, peptides have significant advantages such as low toxicity, weak immunogenicity, high safety, strong specificity, and environmentally friendly production processes. Currently, peptide drugs have demonstrated good clinical efficacy in the treatment of multiple diseases. Based on the above background, through systematic design and screening, we successfully identified peptides with significant inhibitory effects on myocardial fibrosis. This invention provides a novel intervention strategy for the prevention and treatment of myocardial fibrosis. Summary of the Invention

[0006] In view of the above-mentioned technical problems in the prior art, the present invention provides a novel polypeptide and its uses, which aims to solve the technical problem that existing drugs are not effective in treating myocardial fibrosis.

[0007] This invention provides a polypeptide whose amino acid sequence is shown in SEQ ID NO.1.

[0008] Specifically, the sequence of the polypeptide is: MTSRQEDASGKKTTEGAKFQGS.

[0009] The present invention also provides a DNA molecule encoding the above-mentioned polypeptide.

[0010] The present invention also provides a recombinant vector containing the above-described DNA molecule.

[0011] The present invention also provides a host cell comprising the above-described recombinant vector.

[0012] The present invention also provides an expression system containing the above-described recombinant vector, or an exogenous DNA molecule integrated into the genome.

[0013] The present invention also provides the use of the above-mentioned polypeptide in the preparation of a medicament for treating myocardial fibrosis.

[0014] The present invention also provides a pharmaceutical composition, characterized in that its active ingredient is the aforementioned polypeptide.

[0015] Furthermore, the pharmaceutical composition also contains pharmaceutical excipients.

[0016] Furthermore, the dosage form of the pharmaceutical composition is tablets, capsules, or injections.

[0017] Compared to existing peptides for treating myocardial fibrosis, this invention's peptide is secreted in large quantities under physiological conditions following exercise. Therefore, this peptide has no significant adverse effects on the organism. In normal organisms and organisms in the early stages of disease, increasing exercise can alleviate myocardial fibrosis. In the middle and late stages, in vitro administration of this peptide can alleviate myocardial fibrosis, especially under conditions of limited exercise. Experiments have demonstrated that this invention can alleviate myocardial fibrosis.

[0018] Compared with existing technologies, the technical effects of this invention are positive and significant. Compared with existing drugs for treating myocardial fibrosis, this polypeptide has fewer side effects, lower immunogenicity, and lower energy consumption in production. Furthermore, the polypeptide of this invention can cover patients with multiple risk factors, enabling better management and treatment of patients. Attached Figure Description

[0019] Figure 1 The changes observed after drug treatment are shown in the control group: A. Left ventricular ejection fraction in mice; B. Short axis shortening in mice; C. Left ventricular end-diastolic diameter in mice; D. Left ventricular end-systolic diameter in mice; E. Left ventricular end-diastolic volume in mice; F. Left ventricular end-systolic volume in mice; G. Left ventricular mass in mice; H. Systolic blood pressure in mice; I. Diastolic blood pressure in mice.

[0020] Figure 2 The homology of the polypeptides of the present invention in vertebrates is demonstrated. Detailed Implementation

[0021] Example 1: Screening of peptides

[0022] We constructed an endurance exercise mouse model characterized by low-intensity, long-duration training. Using 8-week-old male C57BL / 6 mice as the research subjects, we first conducted adaptive exercise training for 2 days by running for 5 minutes a day at a speed of 10 meters per minute. Then, the mice started running exercise training at an initial speed of 10 meters per minute every day, increasing the speed by 1 meter per minute every 20 minutes until exhaustion. The exercise training lasted for 14 days.

[0023] Normally fed, non-exercised mice served as the control group.

[0024] After modeling, heart tissues from mice in each group were collected for peptidomics analysis. Bioinformatics analysis of the sequencing results was performed based on Log2(fold change) > 1 and P < 0.05, revealing a peptide (Mus_musculus / 1-22) whose expression significantly increased after exercise. The function and mechanism of this peptide have not yet been reported. The peptide encoding this amino acid shows high homology in various vertebrates, including humans, mice, and rats. Figure 2 This demonstrates evolutionary conservation, suggesting that it may have important biological functions.

[0025] The sequence of the polypeptide is: MTSRQEDASGKKTTEGAKFQGS.

[0026] Example 2: Method for preparing polypeptides

[0027] The polypeptide described in Example 1 was prepared using a conventional method, as detailed below:

[0028] The polypeptide is synthesized from the C-terminus to the N-terminus.

[0029] 1. Weigh 3g of RINK resin (degree of substitution 0.3mmol / g) into a 150ml reactor and soak it in 50ml of dichloromethane (DCM).

[0030] 2.2 hours later, wash the resin with 3 times the resin volume of nitrogen-dimethylformamide (DMF), then dry it. Repeat this process four times until the resin is completely dry and ready for use.

[0031] 3. Add a certain amount of 20% piperidine (piperidine / DMF) to the reactor and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting groups on the resin. After deprotection, wash four times with DMF at a volume of 3 times the resin volume, and then dry it.

[0032] 4. Take a small amount of resin and test it using the ninhydrin (Ninhydrin hydrate) method (two drops each of test A and test B, react at 100℃ for 1 min). If the resin turns colored, it indicates that the deprotection was successful.

[0033] 5. Weigh an appropriate amount of the first amino acid at the C-terminus and an appropriate amount of 1-hydroxy-benzotriazole (HOBT) into a 50ml centrifuge tube, add 20ml of DMF to dissolve them, then add 3ml of N,N-diisopropylcarbodiimide (DIC) and shake well for 1min. After the solution becomes clear, add it to the reactor and then place the reactor in a shaker at 30℃ to react.

[0034] 6.2 hours later, end the resin with a certain amount of acetic anhydride (acetic anhydride: DIEA: DCM = 1:1:2) for half an hour, then wash four times with 3 times the volume of DMF, and dry for later use.

[0035] 7. Add a certain amount of 20% piperidine (piperidine / DMF = 1:4) to the reactor, and shake on a decolorizing shaker for 20 minutes to remove the Fmoc protecting groups on the resin. After deprotection, wash four times with DMF and then dry.

[0036] 8. Take a small amount of resin and test it using the ninhydrin (Ninhydrin hydrate) method (two drops each of test A and test B, react at 100℃ for 1 min). If the resin turns colored, it indicates that the deprotection was successful.

[0037] 9. Weigh an appropriate amount of the second amino acid and HOBT into a 50ml centrifuge tube, add 25ml of DMF to dissolve them, then add 2.5ml of DIC and shake well for 1min. After the solution is clear, add it to the reactor and then place the reactor in a shaker at 30℃ to react.

[0038] After 10.1 hours, take a small amount of resin for testing using the ninhydrin method (two drops each of test A and test B, react at 100℃ for 1 min). If the resin is colorless, the reaction is complete; if the resin is colored, the condensation is incomplete, and the reaction should continue.

[0039] 11. After the reaction is complete, wash the resin four times with DMF, then dry it under vacuum. Add a certain amount of 20% piperidine (piperidine / DMF = 1:4) to the reactor and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting groups on the resin. After deprotection, wash the resin four times with DMF, then dry it under vacuum to check whether the protection has been removed.

[0040] 12. Follow steps 9-11 to connect the following amino acids in sequence.

[0041] 13. After the last amino acid is added, the protection is removed, and the resin is washed four times with DMF. Then, the resin is dried with methanol. The peptide is then cleaved from the resin using 95% cleavage buffer (trifluoroacetic acid: 1,2-ethylenedithiol: 3, isopropylsilane: water = 95:2:2:1) (10 ml of cleavage buffer per gram of resin), and centrifuged four times with ice-cold ether (cleavage buffer: ether = 1:9). Finally, the peptide is purified by HPLC and then lyophilized to obtain a peptide of a certain purity.

[0042] 14. The purification conditions are as follows:

[0043] Stationary phase: C18 column (commercially available product);

[0044] Configuration of the mobile phase:

[0045] PumpA: V(tfa) / V(water)=1 / 1000;

[0046] PumpB: V(TFA) / v(acetonitrile) = 1 / 1000;

[0047] Flow rate: 10 ml / min;

[0048] Retention time: 20-30 minutes.

[0049] Example 3 Application of polypeptides

[0050] (1) Experimental animals and grouping:

[0051] Eight-week-old male C57BL / 6 mice were randomly divided into four groups (n=3):

[0052] Control group: Standard feeding with no intervention;

[0053] Polypeptide group: Polypeptide injected via tail vein;

[0054] Ang II Model Group: Ang II Modeling;

[0055] Ang II+ Peptide Group: Ang II modeling + tail vein injection of peptides.

[0056] (2) Model building:

[0057] A myocardial fibrosis model was established by subcutaneous implantation of Ang II micro-osmotic pump in the neck and back at a dose of 1.5 mg / kg / d. The peptide group and the Ang II+ peptide group were injected with peptide-related viruses via tail vein.

[0058] (3) Echocardiography:

[0059] Four weeks after intervention, cardiac function was assessed using a high-resolution small animal ultrasound imaging system (Fujifilm VisualSonics Vevo3100, probe frequency 30 MHz). During the examination, anesthesia was maintained using 1-2% isoflurane at a flow rate of 0.6-1 L / min, and electrocardiograms were monitored simultaneously (heart rate stabilized at 450±25 bpm). Left ventricular mass (LVMass, mg), end-systolic / end-diastolic diameters (D;s, D;d, mm) were measured, and left ventricular end-systolic / end-diastolic volumes (V;s, V;d, μl), ejection fraction (EF%), and fractional shortening (FS%) were calculated.

[0060] (4) Blood pressure measurement:

[0061] Hemodynamic parameters were recorded using a non-invasive tail artery blood pressure measurement system (Softron Biotechnology) for systolic blood pressure (SBP, mmHg) and diastolic blood pressure (DBP, mmHg).

[0062] (5) Results:

[0063] Compared with the control group, the AngII model group mice exhibited typical pathological compensatory characteristics in cardiac function. Regarding cardiac systolic function, the left ventricular ejection fraction (EF%) and fractional shortening (FS%) of the AngII model group mice were significantly reduced. Figure 1 A, Figure 1 B), the end-systolic diameter (D;s) and end-diastolic diameter (D;d) increase, accompanied by an increase in end-diastolic volume (V;d) and end-systolic volume (V;s). Figure 1 C Figure 1 D、 Figure 1 E, Figure 1 F) indicates decreased left ventricular pumping efficiency and impaired myocardial contractility; in terms of structural remodeling, left ventricular mass (LVMass) is significantly increased (F) Figure 1 G); Regarding arterial blood pressure, both systolic blood pressure (SBP) and diastolic blood pressure (DBP) were significantly elevated ( Figure 1 H, Figure 1 I).

[0064] Compared with the Ang II model group, the combined intervention group (Ang II + peptide) significantly improved cardiac pathological processes. The ejection fraction (EF%) and fractional shortening (FS%) in the combined intervention group (Ang II + peptide) were significantly higher than those in the Ang II model group. Figure 1 A, Figure 1 B), the end-systolic diameter (D;s) and end-diastolic diameter (D;d), end-diastolic volume (V;d) and end-systolic volume (V;s) were all smaller than those in the Ang II group. Figure 1 C Figure 1 D、 Figure 1 E, Figure 1 F), indicating improved left ventricular systolic function; in terms of structural remodeling, the left ventricular mass (LVMass) in the combined intervention group (Ang II+ peptide) was significantly reduced ( Figure 1 G), Regarding arterial blood pressure, the combined intervention group (Ang II + peptide) had lower systolic blood pressure (SBP) and diastolic blood pressure (DBP) compared to the Ang II group. Figure 1 H, Figure 1 I), all of the above indicate that peptides can effectively inhibit ventricular dilation and pathological hypertrophy, lower blood pressure, and effectively reduce the risk of heart failure. Figure 1 ).

Claims

1. A polypeptide, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

1.

2. A DNA molecule encoding the polypeptide of claim 1.

3. A recombinant vector, characterized in that, It contains the DNA molecule as described in claim 2.

4. A host cell, characterized in that, It includes the recombinant vector as described in claim 3.

5. An expression system, characterized in that, The expression system contains the recombinant vector of claim 3, or the genome in which an exogenous DNA molecule of claim 2 is integrated.

6. Use of the polypeptide of claim 1 in the preparation of a medicament for treating myocardial fibrosis.

7. A pharmaceutical composition, characterized in that, Its active ingredient is the polypeptide described in claim 1.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition also contains pharmaceutical excipients.

9. A pharmaceutical composition according to claim 7, characterized in that, The dosage form of the pharmaceutical composition is an injection.

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